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Optimized Processing of Growth Factor MobilizedPeripheral Blood CD34 Products by CounterflowCentrifugal Elutriation

CHY-ANH TRAN, MONICA TORRES-CORONADO, AGNES GARDNER, ANGEL GU, HIEU VU, ANITHA RAO,LAN-FENG CAO, AMIRA AHMED, DAVID DIGIUSTO

Key Words. Stem cell CD34 Immunodeficient mouse Cellular therapy

Beckman Research Instituteof the City of Hope, Duarte,California, USA

Correspondence: David DiGiusto,Ph.D., Beckman ResearchInstitute, City of Hope, 1500 EastDuarte Road, Duarte, CA 91010,USA. Telephone: 626-256-4673;Fax: 626-301-8261; e-mail:ddigiusto@coh.org

Received December 15, 2011;accepted for publication April10, 2012; first published onlinein SCTM EXPRESS May 8, 2012.

AlphaMed Press1066-5099/2012/$20.00/0

http://dx.doi.org/10.5966/sctm.2011-0062

ABSTRACT

Cell separation by counterflow centrifugal elutriation has been described for the preparation ofmonocytes for vaccine applications, but its use in other current goodmanufacturing practice (cGMP)operations has been limited. In this study, growth factor-mobilized peripheral blood progenitor cellproducts were collected from healthy donors and processed by elutriation using a commercial cellwashing device. Fractions were collected for each product as per the manufacturers instructions orusing a modified protocol developed in our laboratory. Each fraction was analyzed for cell count,viability, and blood cell differential. Our data demonstrate that, using standard elutriation proce-dures, >99% of red blood cells and platelets were removed from apheresis products with highrecoveries of total white blood cells and enrichment of CD34 cells in two of five fractions. Withmodification of the basic protocol, we were able to collect all of the CD34 cells in a single fraction.The CD34-enriched fractions were formulated, labeled with a ferromagnetic antibody to CD34,washed using the Elutra device, and transferred directly to a magnetic bead selection device forfurther purification. CD34 cell purities from the column were extremely high (98.7 0.9%), andyields were typical for the device (55.7 12.3%). The processes were highly automated and closedfrom receipt of the apheresis product through formulation of target-enriched cell fractions. Thus,elutriation is a feasible method for the initial manipulations associated with primary blood celltherapy products and supports cGMP and current good tissue practice-compliant cellprocessing. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:422429

INTRODUCTION

Hematopoietic stemandprogenitor cells (HSPCs)have been widely used to provide long-lastinghematopoietic reconstitution following ablativetherapy for cancer [18] and in gene therapy ap-plications [915]. However, the inherent plastic-ity in CD34 differentiation and apparent para-crine effects on necrotic or ischemic tissue hasgenerated significant nonhomologous applica-tion of this important cell source. Specifically, theclinical utility of CD34 cells in critical limb isch-emia [1618], chronic liver disease [19], andpostinfarct myocardial recovery [2023] hasbeen widely evaluated. With such expanded useof CD34 cells for cellular therapy, the isolationand enrichment of these cells is of great interestto investigators for both research and clinicaltherapeutic development.

The most readily available source of CD34human HSPCs is granulocyte-colony stimulatingfactor (G-CSF)-mobilized peripheral blood, whichis collected as an apheresis product, hematopoi-etic progenitor cell apheresis (HPC-A). HPC-Aproducts typically contain 1050 109 white

blood cells and more than 50 1010 plateletsand red blood cells. Platelets and red blood cellsmust be removed from these products prior tosubsequentmanipulations designed to enrich fortarget cell populations (e.g., CD34-cell enrich-ment). Additionally, the HPC-A product must bebuffer-exchanged and incubated with antibody-coated magnetic beads (or fluorochrome-conju-gated antibodies), then washed again to removenonbound antibody, and formulated in thespecified amount and type of buffer for cell en-richment over amagnetic cell selection device orfluorescence-activated cell sorter. Typically,washing and formulation procedures are per-formed manually using repeated cycles of cen-trifugation followed by removal of supernatantwith a plasma extractor and dilution in bufferedsaline. This procedure is time-consuming, labor-intensive, and subject to operator-related vari-ability. Moreover, the manipulations required tokeep the system closed (repeated tubing weldsto buffer bags, removal for centrifugation, andthen rewelding for next buffer/wash) increasethe potential for contamination or leakage of the

PROTOCOLS AND MANUFACTURING FOR CELL-BASEDTHERAPIES

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product. Thus, we determined that a more robust system forprocessing HPC-A products was warranted in support of ourgood manufacturing practice and manufacturing operations.

Since it was first introduced in the 1970s [2428], counter-flow centrifugal elutriation (CCE) has been used extensively inresearch applications to separate cell products on the basis ofsize and density. More recently, a clinical elutriation device hasbeen developed (Elutra; GambroBCT, Lakewood, CO, http://www.caridianbct.com) and has been successfully used to isolatemonocytes fromperipheral blood apheresis products for vaccineapplications [2936] and lymphocytes for adoptive immuno-therapy [37]. During elutriation, platelets and red blood cells areefficiently separated from white blood cells with monocyteshighly enriched in a single fraction. On the basis of these results,we began an evaluation for the use of the Elutra system as ageneral tool for preparation of HPC-A products for downstreamprocessing. Our results support the implementation of this auto-mated approach for HSPC isolation in most cell-processing labo-ratories.

MATERIALS AND METHODS

Starting MaterialHPC-A products were obtained from AllCells LLC (Emeryville, CA,http://www.allcells.com), Key Biologics LLC (Memphis, TN,http://www.keybiologics.com), or Progenitor Cell Therapy LLC(Mountain View, CA, http://www.pctcelltherapy.com). HPC-Aproducts were collected from healthy adults following 34 daysof G-CSF (510g/kg/day) mobilization and processed within 24hours of collection. Informed consent was obtained for each do-nor by individual vendors according to vendor-specific protocolsand institutional review board review.

Cell CountsWhite blood cell (WBC), red blood cell (RBC), and platelet countswere obtained using an AcT 5diff CP Hematology Analyzer (Beck-man Coulter, Brea, CA, http://www.beckmancoulter.com) ac-cording to the manufacturers protocol. Cell viability was deter-mined using the Guava Viacount Assay (Guava Technologies,Hayward, CA, http://www.guavatechnologies.com) in accor-dance with the manufacturers recommendations.

ElutriationThe disposable Elutra tubing set is presterilized and is a function-ally closed system that provides a means to individually connectblood, buffer, waste, and final product bags to a spinning cellseparation chamber. Each Elutra set can process up to 400 ml ofstarting product, provided that cell count does not exceed 3 1010 WBCs or 7.5 ml of RBCs. Elutra protocol 1 is the standardmanufacturers protocol that separates cells into five fractionsand is intended for enrichment of monocytes in fraction 5. Frac-tion 1 is collected using a pump speed of 37 ml/minute, 2,400rpm. Fraction 2 is collected using a pump speed of 68ml/minute,2,400 rpm in approximately 975 ml. Fraction 3 is collected usinga pump speed of 74ml/minute, 2,400 rpm in 975ml. Fraction 4 iscollected using a pump speed of 103 ml/minute, 2,400 rpm in975 ml. Fraction 5 is collected using a pump speed of 125 ml/minute at 0 rpm in approximately 300 ml. Elutra protocol 2 wasdeveloped in our laboratory in collaboration with the manufac-turer and elutriates cells into three fractions. Fraction 1 is col-

lected using a pump speed of 60 ml/minute, 2,400 rpm in ap-proximately 1,000ml. Fraction 2 is collected using a pump speedof 25 ml/minute, 2,400 rpm in 450 ml. Fraction 3 is collected bystopping the rotor (0 rpm) and pumping at 5ml/minute to a finalvolume of 5080 ml.

The Elutra was also used to wash the bead-labeled cells inconjunctionwith protocol 2. After bead labeling, the cell bagwasreconnected to the original Elutra disposable set. Cells weretransferred using a cell inlet flow rate of 10 ml/minute, 2,400rpm using 200 ml of a CliniMACS (Miltenyi Biotec, BergischGladbach, Germany, http://www.miltenyibiotec.com) ethylene-diaminetetraacetic acid (EDTA) phosphate-buffered saline(PBS) 0.5% human serum albumin (HSA) buffer. An additional500-ml buffer was used to wash the cells with a gradual increasein flow rate of 2 ml/minute every 2 minutes to a maximum of 25ml/minute. Then, the cell inlet flow rate was set to 0 ml/minuteand rotor speed adjusted to 2,000 rpm to sediment the cells. Thecells were then transferred to the collection bag at 5 ml/minute.Once all cells were removed from the chamber and the outletline, the debulk pump flow rate was set to 25ml/minute and themedium pump flow rate to 30 ml/minute to clear the line of anyresidual cells. The cells were collected in a maximum volume of120 ml for loading onto the CliniMACS device.

Bag Wash by CentrifugationG-CSF-mobilized apheresis products were diluted with three vol-umes of CliniMACS buffer 0.5% HSA before magnetic labeling.Cells were pelleted by centrifugation at 200g, room tempera-ture, acceleration 9, and deceleration 2 for 15 minutes. Samplesfor analysis were taken after an additional wash followingCD34 microbead labeling.

CD34 SelectionFor CD34 e